Holmes D L, Stellwagen N C
Department of Biochemistry, University of Iowa, Iowa City 52242.
Electrophoresis. 1990 Jan;11(1):5-15. doi: 10.1002/elps.1150110103.
The electric field dependence of the electrophoretic mobility of linear DNA fragments in agarose gels was reinvestigated in order to correct the observed mobilities for the different temperatures actually present in the gel during electrophoresis in different electric field gradients. When corrected to a common temperature, the electrophoretic mobilities of DNA fragments less than or equal to 1 kilobase pairs (kbp) in size were independent of electric field strength at all field strengths from 0.6 to 4.6 V/cm if the gels contained less than or equal to 1.4% agarose. The mobilities of larger DNA fragments increased approximately linearly with electric field strength. If the agarose concentration was higher than 2%, the mobilities of all DNA fragments increased with increasing electric field strength. The electric field dependence of the mobility was larger in gels cast and run in Tris-borate buffer (TBE) than in gels cast and run in Tris-acetate buffer (TAE), and was more pronounced in gels without ethidium bromide incorporated in the matrix. Ferguson plots were constructed for the various DNA fragments, both with and without extrapolating the temperature-corrected mobilities to zero electric field strength. Linear Ferguson plots were obtained for all fragments less than or equal to 12 kbp in size in agarose gels less than or equal to 1.4% in concentration if the mobilities were first extrapolated to zero electric field strength. Concave upward curvature of the Ferguson plots was observed for DNA fragments greater than or equal to 2 kbp in size at finite electric field strengths. Convex downward curvature of the Ferguson plots was observed for DNA fragments greater than or equal to 1 kbp in size in agarose gels greater than or equal to 2% in concentration. The mobilities of the various DNA fragments, extrapolated to zero agarose concentration and zero electric field strength, decreased with increasing DNA molecular weight; extrapolating to zero molecular weight gave an "intrinsic" DNA mobility of 2.7 x 10(-4) cm2/Vs at 20 degrees C. The pore sizes of LE agarose gels cast and run in TAE and TBE buffers were estimated from the mobility of the DNA fragments.(ABSTRACT TRUNCATED AT 400 WORDS)
为了校正线性DNA片段在琼脂糖凝胶中电泳迁移率的电场依赖性，对不同电场梯度电泳过程中凝胶内实际存在的不同温度下所观察到的迁移率进行了重新研究。当校正到一个共同温度时，如果凝胶中琼脂糖含量小于或等于1.4%，那么大小小于或等于1千碱基对（kbp）的DNA片段的电泳迁移率在0.6至4.6V/cm的所有场强下均与电场强度无关。较大DNA片段的迁移率随电场强度近似呈线性增加。如果琼脂糖浓度高于2%，所有DNA片段的迁移率都随电场强度增加而增加。在Tris-硼酸缓冲液（TBE）中制备和运行的凝胶中，迁移率的电场依赖性比在Tris-乙酸缓冲液（TAE）中制备和运行的凝胶中更大，并且在基质中未掺入溴化乙锭的凝胶中更为明显。针对各种DNA片段构建了弗格森图，包括将温度校正后的迁移率外推至零电场强度和不进行外推的情况。如果首先将迁移率外推至零电场强度，对于浓度小于或等于1.4%的琼脂糖凝胶中大小小于或等于12kbp的所有片段，可获得线性弗格森图。在有限电场强度下，对于大小大于或等于2kbp的DNA片段，观察到弗格森图向上凹的曲率。对于浓度大于或等于2%的琼脂糖凝胶中大小大于或等于1kbp的DNA片段，观察到弗格森图向下凸的曲率。将各种DNA片段的迁移率外推至零琼脂糖浓度和零电场强度时，其迁移率随DNA分子量增加而降低；外推至零分子量时，在20℃下得到“固有”DNA迁移率为2.7×10⁻⁴cm²/Vs。根据DNA片段的迁移率估算了在TAE和TBE缓冲液中制备和运行的低熔点琼脂糖凝胶的孔径。（摘要截断于400字）
